Notch2 controls developmental fate choices between germinal center and marginal zone B cells upon immunization

Sustained Notch2 signals induce trans-differentiation of Follicular B (FoB) cells into Marginal Zone B (MZB) cells in mice, but the physiology underlying this differentiation pathway is still elusive. Here, we demonstrate that most B cells receive a basal Notch signal, which is intensified in pre-MZB and MZB cells. Ablation or constitutive activation of Notch2 upon T-cell-dependent immunization reveals an interplay between antigen-induced activation and Notch2 signaling, in which FoB cells that turn off Notch2 signaling enter germinal centers (GC), while high Notch2 signaling leads to generation of MZB cells or to initiation of plasmablast differentiation. Notch2 signaling is dispensable for GC dynamics but appears to be re-induced in some centrocytes to govern expansion of IgG1+ GCB cells. Mathematical modelling suggests that antigen-activated FoB cells make a Notch2 dependent binary fate-decision to differentiate into either GCB or MZB cells. This bifurcation might serve as a mechanism to archive antigen-specific clones into functionally and spatially diverse B cell states to generate robust antibody and memory responses.


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Life sciences study design
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Sample size
Sample sizes were determined based on previous experiments and preliminary data.No statistical method was used to predetermine sample size.Our goal was to analyze at least 6 animals per group.In cases, in which differences were pronounced enough to achieve significance with smaller sample sizes, adjustments were made accordingly.In general, we analyzed a minimum of 4 samples per genotype and treatment.
There were some exceptions.In Fig. 4a (5h time point), the sample size was n=2 and was excluded from the statistical analysis.In Fig. 7c, Supplementary Fig. 2c, Supplementary Fig. 4, ELISpots in supplementary Fig. 7a n=3 mice were analysed.In most cases, the data presented in these figures are from experiments performed during the first revision phase.During this time, we only had a limited number of mice available and were therefore unable to perform all the experiments requested by the reviewers with higher numbers of mice.Despite the smaller number of mice, our analyses still yielded significant results or, at least, clear trends, as the biological differences between the samples were sufficiently robust.
For mathematical modeling, values were collected from at least two independent immunization rounds, with the exception of day 17, where one immunization round with 3 mice was performed.At each time point, a minimum of 3 mice per genotype were utilized for data gathering.
Data exclusions No data exclusions were done.

Replication
All in vivo experiments were performed in at least two or more independent immunization rounds.In vitro experiments were performed at least in duplicates with the indicated numbers of mice.Replications of in vivo and in vitro experiments were successful.
Randomization For all in vivo and in vitro studies, mice were allocated in cohorts based on genotype and/or time points after NP-CGG antigen injection (immunization) and no randomization was performed.Mice used in the study were maintained at minimally perturbative conditions.Control and analytical mice were always analysed in parallel and predominantly used in an age-matched manner, when possible.The experiments were repeated with different individual animals and several rounds of independent immunizations in several experiments.

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April 2023

Blinding
In all experiments, the investigators were not blinded, as phenotype observations in flow cytometry, ELISA/ELISpot and in microscopy were clearly visible and consistent with the genotype of the analytical animal(s).Flow cytometry data analyses were performed unbiased, with consistent gating of lymphocyte populations for all genotypes and time points.

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Wild animals
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Reporting on sex
Findings from this study do not apply to only one sex.Sex of mice was not considered as a decisive factor in the study design and methods.Both male and female mice were used for the experiments, data were analyzed without consideration of the animals' sex.
To ensure a more balanced distribution of both male and female animals throughout the study, control and analytical mice were sexmatched in experiments, whenever possible.
A detailed overview of the sex and age of each mouse is provided in the source data file (Excel) for each Figure showing experimental data with mice.
Field-collected samples N/A

Ethics oversight
The experiments were performed in compliance with the German Animal Welfare Law and were approved by the Institutional Committee on Animal Experimentation and the government of Upper Bavaria.
Note that full information on the approval of the study protocol must also be provided in the manuscript.The axis labels state the marker and fluorochrome used (e.g.CD4-FITC).

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The axis scales are clearly visible.Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology Sample preparation
Single cell suspensions were prepared from the spleen and bone marrow (BM) of mice.Following organ extraction, the SP were preserved in 1% B cell medium on ice (BCM; 1x RPMI 1640 supplemented with 1% (v/v) heat-inactivated fetal calf serum (FCS) (PAA Cell culture Company), 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, 2 mM Lglutamine, 1x non-essential amino acids, and 50 μM β-mercaptoethanol (all purchased from Gibco)).BM was collected by flushing the femur and tibia bones with 1% BCM and a cannula.For the preparation of single cell suspensions, SP was mashed through a 70 μM strainer.Following centrifugation at 1200 rpm for 10 min at 4 degrees, spleen and BM cell suspensions were lysed for 3 min at room temperature in 1 mL of red blood lysis buffer (RBC) buffer (1x, eBioscience) to remove erythrocytes.The reaction was terminated by addition of 12 mL 1% BCM and centrifugation under the same conditions.Finally, cells from the spleen and BM were resuspended in 1 mL 1% BCM and were counted to establish the cell number for Flow Cytometry experiments.Surface staining of lymphocytes with the corresponding antibodies in MACS buffer (Miltenyi) was performed on ice for 25 min.To omit dead cells from the analysis, cells were stained for 20 min on ice with LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Invitrogen) prior to extracellular antibody staining.

Cell population abundance
For the in-vitro experiment measuring Notch2 surface expression on purified FoB cells, the FoB cells were isolated with the MZB and FoB Cell Isolation Kit (Miltenyi Biotech).The enrichment of mature B cells was checked in a preliminary staining prior to the experiment by a CD19 vs. Thy1.2co-staining (purified B cells were identified as CD19+ Thy1.2-, data not shown here).The purity of FoB cells (no MZB cell contamination) was reassured in a subsequent staining for CD23 and CD21 within the CD19+ Thy1.2-cell population (preliminary staining, data not shown).FoB cells were herein identified as CD23+ CD21low/ neg.The purity of the used FoB cells was always at least 96%.
For the plasmablast differentiation in vitro assay, total naïve B cells were isolated from splenic cell suspensions with the CD43 depletion B cell Isolation Kit (Miltenyi).The enrichment and purity of B cells was ensured in a CD19 vs. CD43 surface staining (B cells were identified as CD19+CD43-, pre-experiment staining for purity, data not shown).The purity of B cells reached >95% for all experiments.

Gating strategy
Representative gating strategies (refering to most flow cytometry data in this manuscript) for surface and intracellular cell stainings to distinguish single living B lymphocytes and their subpopulations, reporter-expressing and reporter-negative cells, as well as plasmablasts and plasma cells live lymphocytes, have been provided in Supplementary Figure 14a-e.
Otherwise important gating strategies to identify specific B cell subpopulations (e.g.GC B cells, IgG1-switched cells, light and dark zone GC B cells etc.) are always shown in the contour or pseudocolor plots in each figure containing flow cytometry analyses.In addition, detailed information about the proper identification of cells using their differential extracellular or intracellular marker expression are described in all figure legends.For the mathematical modelling part of the manuscript, specific sequential gating of cell subsets was used.The full hierarchical gating strategy for this part is therefore shown in a separate Supplementary Figure 11.
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.
If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.Male and female mice at the age of 10-18 weeks were used for all experiments.All mice were on a pure Balb/c background, except for CBF:H2B-Venus reporter mice, which were backcrossed after purchase for 5 generations on a Balb/c background.All animal experiments were performed in compliance with the German Animal Welfare Law and were approved by the Institutional Committee on Animal Experimentation and the Government of Upper Bavaria.The animals were kept in the laboratory animal rooms under specified pathogen-free (SPF) conditions with a 12/12-hour light cycle.The animal rooms are fully air-conditioned, the set-points are set to 20 -24°C temperature and 45 -65 % humidity according to Annex A of the European Convention 2007/526 EC.The rooms are equipped with individually ventilated cage systems (e.g.Tecniplast Greenline GM 500, 501 cm² floor area, or BioZone-IVC type II, 370 cm² floor area).The maximum stocking densities comply with Annex III of Directive 2010/63/EU.The cages are equipped with laboratory animal bedding (wood fiber/wood chips, e.g.Lignocel Select Fine, SAFE).The animals were given sterile filtered water and a standard rodent diet (e.g.Altromin 1314) ad libitum.Control and experimental mice were bred separately, but were maintained in the same area of the mouse facility.The mice were euthanized either with CO2 or by cervical dislocation.The QuietekTM-apparatus was used for euthanasia with CO2 to ensure that the CO2 was supplied with the correct flow rate up to the correct filling volume.
For intracellular staining, cells were fixed with 2% paraformaldehyde (1:2 Carl Roth) for 10 min at room temperature and permeabilized in ice-cold 100% methanol for 10 min on ice.Cells were incubated for 1 h at room temperature in the dark with the corresponding antibodies.Dead cells were excluded by staining for 5 min on ice with LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Invitrogen) before fixation.